PROBING THE MOLTEN GLOBULE STATE OF ALPHA-LACTALBUMIN BY LIMITED PROTEOLYSIS

Limited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N-terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross-linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis. Considering the broad substrate specificity of both pepsin and thermolysin and, thus, the very numerous potential sites of proteolytic attack along the 123-residue chain of BLA, these results indicate that BLA in its partially folded A- or TFE-state is a more dynamic entity than in the native state, but is still a structured and relatively rigid protein, preventing extensive degradation by proteolysis. All sites of limited proteolysis observed in this study are located outside the helical chain segments of BLA in its native or partially folded states [Alexandrescu, A. T., Ng, Y.-L., & Dobson, C. M. (1994) J. Miol. Biol. 235, 587-599]. The fast initial nicking at chain segment 40-53 of BLA by both pepsin and thermolysin indicates that this region in both the A- and TFE-states of BLA are characterized by the exposure and flexibility required for a productive interaction at the active site of the attacking protease. Indeed, previous NMR measurements have indicated that the single beta-sheet region (chain segment 40-53) of the native structure, at variance from the helical segments, is not presented in the A- or TFE-state of BLA.